Dna-binding protein using ppr motif, and use thereof

ABSTRACT

The object of the present invention is to, by analyzing PPR proteins that act to bind to DNA with a prediction that RNA recognition rules of PPR motifs can also be used for recognition of DNA, find a PPR protein showing such a characteristic. According to the present invention, it was revealed that, with a protein that can bind in a DNA base-selective manner or a DNA base sequence-specific manner, which contains one or more, preferably 2 to 30, more preferably 5 to 25, most preferably 9 to 15, of PPR motifs having a structure of the following formula 1 (wherein, in the formula 1, Helix A is a part that can form an α-helix structure; X does not exist, or is a part consisting of 1 to 9 amino acids; Helix B is a part that can form an α-helix structure; and L is a part consisting of 2 to 7 amino acids), and having a specific combination of amino acids corresponding to a DNA base or DNA base sequence as amino acids of three positions of No. 1 A.A., No. 4 A.A., in Helix A of the formula 1 and No. “ii” (-2) A.A. contained in L of the formula 1, the aforementioned object could be achieved.(Helix A)-X-(Helix B)-L  (Formula 1)

CROSS-REFERENCE OF RELATED APPLICATIONS

This application is a Divisional of application Ser. No. 16/216,617,filed Dec. 11, 2018, which is a Divisional of application Ser. No.14/785,952, filed Oct. 21, 2015, which is a 371 of InternationalApplication No. PCT/JP2014/061329, filed Apr. 22, 2014, which claimspriority of Japanese Patent Application No. 2013-089840, filed Apr. 22,2013, the entire contents of which are incorporated herein by reference.

TECHNICAL FIELD

The present invention relates to a protein that can selectively orspecifically bind to an intended DNA base or DNA sequence. According tothe present invention, a pentatricopeptide repeat (PPR) motif isutilized. The present invention can be used for identification anddesign of a DNA-binding protein, identification of a target DNA of aprotein having a PPR motif, and functional control of DNA. The presentinvention is useful in the fields of medicine, agricultural science, andso forth. The present invention also relates to a novel DNA-cleavingenzyme that utilizes a complex of a protein containing a PPR motif and aprotein that defines a functional region.

BACKGROUND ART

In recent years, techniques of binding nucleic acid-binding proteinfactors elucidated through various analyses to an intended sequence havebeen established, and they are coming to be used. Use of thissequence-specific binding is enabling analysis of intracellularlocalization of a target nucleic acid (DNA or RNA), elimination of atarget DNA sequence, or expression control (activation or inactivation)of a protein-encoding gene existing downstream of a target DNA sequence.There are being conducted researches and developments using the zincfinger protein (Non-patent documents 1 and 2), TAL effecter (TALE,Non-patent document 3, Patent document 1), and CRISPR (Non-patentdocuments 4 and 5) as protein factors that act on DNA as materials forprotein engineering. However, types of such protein factors are stillextremely limited.

For example, the artificial enzyme, zinc finger nuclease (ZFN), known asan artificial DNA-cleaving enzyme, is a chimera protein obtained bybinding a part that is constituted by linking 3 to 6 zinc fingers thatspecifically recognize a DNA consisting of 3 or 4 nucleotides and bindto it, and recognizes a nucleotide sequence in a sequence unit of 3 or 4nucleotides with one DNA cleavage domain of a bacterial DNA-cleavingenzyme (for example, FokI) (Non-patent document 2). In such a chimeraprotein, the zinc finger domain is a protein domain that is known tobind to DNA, and it is based on the knowledge that many transcriptionfactors have the aforementioned domain, and bind to a specific DNAsequence to control expression of a gene. By using two of ZFNs eachhaving three zinc fingers, cleavage of one site per 70 billionnucleotides can be induced in theory.

However, because of the high cost required for the production of ZFNs,etc., the methods using ZFNs have not come to be widely used yet.Moreover, functional sorting efficiency of ZFNs is bad, and it issuggested that the methods have a problem also in this respect.Furthermore, since a zinc finger domain consisting of n of zinc fingerstends to recognize a sequence of (GNN)n, the methods also have a problemthat degree of freedom for the target gene sequence is low.

An artificial enzyme, TALEN, has also been developed by binding aprotein consisting of a combinatory sequence of module parts that canrecognize every one nucleotide, TAL effecter (TALE), with a DNA cleavagedomain of a bacterial DNA-cleaving enzyme (for example, FokI), and it isbeing investigated as an artificial enzyme that can replace ZFNs(Non-patent document 3). This TALEN is an enzyme generated by fusing aDNA binding domain of a transcription factor of a plant pathogenicXanthomonas bacterium, and the DNA cleavage domain of the DNArestriction enzyme FokI, and it is known to bind to a neighboring DNAsequence to form a dimer and cleave a double strand DNA. Since, as forthis molecule, the DNA binding domain of TALE found from a bacteriumthat infects with plants recognize one base with a combination of aminoacids at two sites in the TALE motif consisting of 34 amino acidresidues, it has a characteristic that binding property for a target DNAcan be chosen by choosing the repetitive structure of the TALE module.TALEN using the DNA binding domain that has such a characteristic asmentioned above has a characteristic that it enables introduction ofmutation into a target gene, like ZFNs, but the significant superioritythereof to ZFNs is that degree of freedom for the target gene(nucleotide sequence) is markedly improved, and the nucleotide to whichit binds can be defined with a code.

However, since the total conformation of TALEN has not been elucidated,the DNA cleavage site of TALEN has not been identified at present.Therefore, it has a problem that cleavage site of TALEN is inaccurate,and is not fixed, compared with ZFNs, and it also cleaves even a similarsequence. Therefore, it has a problem that a nucleotide sequence cannotbe accurately cleaved at an intended target site with a DNA-cleavingenzyme. For these reasons, it is desired to develop and provide a novelartificial DNA-cleaving enzyme free from the aforementioned problems.

On the basis of genome sequence information, PPR proteins (proteinshaving a pentatricopeptide repeat (PPR) motif) constituting a big familyof no less than 500 members only for plants have been identified(Non-patent document 6). The PPR proteins are nucleus-encoded proteins,but are known to act on or involved in control, cleavage, translation,splicing, RNA edition, and RNA stability chiefly at an RNA level inorganelles (chloroplasts and mitochondria) in a gene-specific manner.The PPR proteins typically have a structure consisting of about 10contiguous 35-amino acid motifs of low conservativeness, i.e., PPRmotifs, and it is considered that the combination of the PPR motifs isresponsible for the sequence-selective binding with RNA. Almost all thePPR proteins consist only of repetition of about 10 PPR motifs, and anydomain required for exhibiting a catalytic action is not found in manycases. Therefore, it is considered that the PPR proteins are essentiallyRNA adapters (Non-patent document 7).

In general, binding of a protein and DNA, and binding of a protein andRNA are attained by different molecular mechanisms. Therefore, aDNA-binding protein generally does not bind to RNA, whereas anRNA-binding protein generally does not bind to DNA. For example, in thecase of the pumilio protein, which is known as an RNA-binding factor,and can encode RNA to be recognized, binding thereof to DNA has not beenreported (Non-patent documents 8 and 9).

However, in the process of investigating properties of various kinds ofPPR proteins, it became clear that it could be suggested that some typesof the PPR proteins worked as DNA-binding factors.

The wheat p63 is a PPR protein having 9 PPR motifs, and it is suggestedby gel shift assay that it binds to DNA in a sequence-specific manner(Non-patent document 10).

The GUN1 protein of Arabidopsis thaliana has 11 PPR motifs, and it issuggested by pull down assay that it binds with DNA (Non-patent document11).

It has been demonstrated by run-on assay that the Arabidopsis thalianapTac2 (protein having 15 PPR motifs, Non-patent document 12) andArabidopsis thaliana DG1 (protein having 10 PPR motifs, Non-patentdocument 12) directly participate in transcription for generating RNA byusing DNA as a template, and they are considered to bind to DNA.

An Arabidopsis thaliana strain deficient in the gene of GRP23 (proteinhaving 11 PPR motifs, Non-patent document 14) shows the phenotype ofembryonal death. It has been demonstrated that this protein physicallyinteracts with the major subunit of the eukaryotic RNA transcriptionpolymerase 2, which is a DNA-dependent RNA transcription enzyme, andtherefore it is considered that GRP23 also acts to bind to DNA.

However, bindings of these PPR proteins to DNA have been only indirectlysuggested, and actual sequence-specific binding has not been fullyverified. Moreover, even if such proteins bind with DNA, it is generallyconsidered that binding of a protein and DNA, and binding of a proteinand RNA are attained by different molecular mechanisms, and thereforewhat kind of sequence rule specifically exists, with which binding isattained, etc., are not even expected at all.

PRIOR ART REFERENCES Patent Documents

-   Patent document 1: WO2011/072246-   Patent document 2: WO2011/111829

Non-Patent Documents

-   Non-patent document 1: Maeder, M. L., et al. (2008) Rapid    “open-source” engineering of customized zinc-finger nucleases for    highly efficient gene modification, Mol. Cell 31, 294-301-   Non-patent document 2: Urnov, F. D., et al. (2010) Genome editing    with engineered zinc finger nucleases, Nature Review Genetics, 11,    636-646-   Non-patent document 3: Miller, J. C., et al. (2011) A TALE nuclease    architecture for efficient genome editing, Nature Biotech., 29,    143-148-   Non-patent document 4: Mali P., et al. (2013) RNA-guided human    genome engineering via Cas9, Science, 339, 823-826-   Non-patent document 5: Cong L., et al. (2013) Multiplex genome    engineering using CRISPR/Cas systems, Science, 339, 819-823-   Non-patent document 6: Small, I. D. and Peeters, N. (2000) The PPR    motif —a TPR-related motif prevalent in plant organellar proteins,    Trends Biochem. Sci., 25, 46-47-   Non-patent document 7: Woodson, J. D., and Chory, J. (2008)    Coordination of gene expression between organellar and nuclear    genomes, Nature Rev. Genet., 9, 383-395-   Non-patent document 8: Wang, X., et al. (2002) Modular recognition    of RNA by a human pumilio-homology domain, Cell, 110, 501-512-   Non-patent document 9: Cheong, C. G, and Hall and T. M. (2006)    Engineering RNA sequence specificity of Pumilio repeats, Proc. Natl.    Acad. Sci. USA 103, 13635-13639-   Non-patent document 10: Ikeda T. M. and Gray M. W. (1999)    Characterization of a DNA-binding protein implicated in    transcription in wheat mitochondria, Mol. Cell Bio., 119 (12):    8113-8122-   Non-patent document 11: Koussevitzky S., et al. (2007) Signals from    chloroplasts converge to regulate nuclear gene expression, Science,    316:715-719-   Non-patent Document 12: Pfalz J, et al. (2006) PTAC2, -6, and -12    are components of the transcriptionally active plastid chromosome    that are required for plastid gene expression, Plant Cell 18:176-197-   Non-patent document 13: Chi W, et al. (2008) The pentatricopeptide    repeat protein DELAYED GREENING1 is involved in the regulation of    early chloroplast development and chloroplast gene expression in    Arabidopsis, Plant Physiol., 147:573-584-   Non-patent document 14: Ding Y H, et al. (2006) Arabidopsis    GLUTAMINE-RICH PROTEIN 23 is essential for early embryogenesis and    encodes a novel nuclear PPR motif protein that interacts with RNA    polymerase II subunit III, Plant Cell, 18:815-830

SUMMARY OF THE INVENTION Object to be Achieved by the Invention

The inventors of the present invention expected that the properties ofthe PPR proteins (proteins having a PPR motif) as RNA adapters would bedetermined by property of each PPR motif constituting the PPR proteinsand combination of a plurality of PPR motifs, and proposed methods formodifying RNA-binding proteins using such PPR motifs (Patent document2). Then, they elucidated that a PPR motif and RNA bind in one-to-onecorrespondence, contiguous PPR motifs recognize contiguous RNA bases inan RNA sequence, and such RNA recognition is determined by combinationof amino acids at specific three positions among the 35 amino acidsconstituting the PPR motif, and filed a patent application for a methodfor designing a customized RNA-binding protein utilizing RNA recognitioncodes of PPR motifs and use thereof (PCT/JP2012/077274; Yagi, Y., et al.(2013) PLoS One, 8, e57286; and Barkan, A., et al. (2012) PLoS Genet.,8, e1002910).

It has been generally considered that binding of a protein and DNA, andbinding of a protein and RNA are attained by different molecularmechanisms. However, the inventors of the present invention predictedthat the RNA recognition rule of the PPR motif would be also usable forrecognition of DNA, and analyzed PPR proteins that act to bind with DNAaiming at retrieving PPR proteins having such a characteristic. Theyalso aimed at providing a novel artificial enzyme by preparing acustomized DNA-binding protein that binds to a desired sequence usingsuch a PPR protein that specifically binds to a DNA obtained asdescribed above, and using it with a protein that defines a functionalregion, and providing a novel artificial DNA-cleaving enzyme by using ittogether with a region having a DNA-cleaving activity as the functionalregion.

Means for Achieving the Object

As for the PPR proteins, it was elucidated by various domain searchprograms (Pfam, Prosite, Interpro, etc.) that the PPR motifs containedin the common RNA-binding type PPR proteins and the PPR motifs containedin the DNA-binding PPR proteins of some kinds mentioned above are notparticularly distinguished. Therefore, it was considered that PPRproteins might contain amino acids (amino acid group) that woulddetermine a binding property for DNA or a binding property for RNA apartfrom the amino acids required for the nucleic acid recognition.

The inventors of the present invention elucidated that an RNA-bindingPPR motif and RNA bind in one-to-one correspondence, contiguous PPRmotifs recognize contiguous RNA bases in an RNA sequence, and in suchrecognition, base-selective binding with RNA is determined bycombination of RNA recognition amino acids at specific three positions(that is, the first and fourth amino acids of the first helix (Helix A)among the two α-helix structures constituting the motif (No. 1 A.A. andNo. 4 A.A.), and the second amino acid counted from the C-terminus (No.“ii” (-2) A.A.)), among the 35 amino acids constituting the PPR motif,and filed a patent application for a method for designing a customizedRNA-binding protein utilizing RNA recognition codes of PPR motifs anduse thereof (PCT/JP2012/077274).

Then, among the PPR proteins, for the aforementioned wheat p63(Non-patent document 11, the amino acid sequence of the homologousprotein of Arabidopsis thaliana is shown as SEQ ID NO: 1), GUN1 proteinof Arabidopsis thaliana (Non-patent document 12, amino acid sequencethereof is shown as SEQ ID NO: 2), pTac2 of Arabidopsis thaliana(Non-patent document 13, amino acid sequence thereof is shown as SEQ IDNO: 3), DG1 (Non-patent document 14, amino acid sequence thereof isshown as SEQ ID NO: 4), and GRP23 of Arabidopsis thaliana (Non-patentdocument 15, amino acid sequence thereof is shown as SEQ ID NO: 5), forwhich binding with DNA was suggested, amino acid frequencies of theamino acids at three positions bearing the nucleic acid recognitioncodes in the PPR motif considered to be important when RNA is a target(No. 1 A.A., No. 4 A.A. and No. “ii” (-2) A.A.) were compared with thosefound in the RNA binding type motif. As a result, it became clear thatthe tendencies of the amino acid frequencies found in those PPR motifsas mentioned above, for which DNA-binding property was suggested, andthe RNA binding type motifs substantially agreed with each other.

The above results suggest that the nucleic acid recognition codes of theRNA binding type PPR motifs can also be applied to the DNA binding typePPR motifs. Thymine (T) is a uracil (U) derivative having a structureconsisting of uracil (U) of which carbon of the 5-position ismethylated, as it is also called 5-methyluracil. Such a characteristicof the base constituting the nucleic acid suggests that the combinationof the amino acids that recognizes uracil (U) of an RNA binding type PPRmotif is used for recognition of thymine (T) in DNA.

On the basis of the aforementioned findings, it was elucidated that, byusing the aforementioned p63 (amino acid sequence of SEQ ID NO: 1), GUN1protein of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 2),pTac2 of Arabidopsis thaliana (amino acid sequence of SEQ ID NO: 3), DG1(amino acid sequence of SEQ ID NO: 4), and GRP23 of Arabidopsis thaliana(amino acid sequence of SEQ ID NO: 5), which are DNA-binding type PPRproteins, as a template, arranging amino acids of the three positions(No. 1 A.A., No. 4 A.A. and No. “ii” (-2) A.A.) with applying thefinding obtained for such PPR proteins as a result of examination of theRNA-binding type PPR motifs, a customized DNA-binding protein that bindsto an arbitrary DNA base sequence could be produced.

That is, the inventors of the present invention provided a protein thatcomprises 2 or more, preferably 2 to 30, more preferably 5 to 25, mostpreferably 9 to 15, of PPR motifs having the specific amino acidsdescribed later as the amino acids at the three positions (No. 1 A.A.,No. 4 A.A., and No. “ii” (-2) A.A.) in the PPR motifs, and can bind toDNA in a DNA base-selective manner or DNA base sequence-selectivemanner, of which typical examples are the amino acid sequences of SEQ IDNOS: 1 to 5, and thus accomplished the present invention.

The present invention provides the followings.

[1] A method for modifying a genetic substance of a cell, the methodincluding: designing a DNA binding protein; determining a DNA basesequence coding for an amino acid sequence of a fused protein comprisinga functional region and a DNA binding region consisting of a DNA-bindingprotein; cloning said DNA base sequence; preparing a vector carryingsaid DNA base sequence and a cell containing a DNA having at least 9target DNA bases as a target DNA base sequence; and introducing thevector into the cell so that the DNA binding region of the fused proteinbinds to the DNA having the at least 9 target DNA bases, and thereforethe functional region modifies the DNA having the at least 9 target DNAbases, wherein the DNA-binding protein contains at least 9 PPR motifsfor binding to the at least 9 target DNA bases, respectively, and havinga structure of the following formula 1: [Formula 1]

(Helix A)-X-(Helix B)-L  (Formula 1)

(wherein, in the formula 1:Helix A is a part that can form an α-helix structure;X does not exist, or is a part consisting of 1 to 9 amino acids;Helix B is a part that can form an α-helix structure; andL is a part consisting of 2 to 7 amino acids),wherein,position numbers of amino acids in the PPR motifs are the same asPF01535 in Pfam under the following definitions:the first amino acid of Helix A is referred to as No. 1 amino acid (No.1 A.A.), the fourth amino acid as No. 4 amino acid (No. 4 A.A.), and

when a next PPR motif (M_(n+1)) contiguously exists on the C-terminusside of the PPR motif (M_(n)) (when there is no amino acid insertionbetween the PPR motifs), the -2nd amino acid counted from the end(C-terminus side) of the amino acids constituting the PPR motif (M_(n));

when a non-PPR motif consisting of 1 to 20 amino acids exists betweenthe PPR motif (M_(n)) and the next PPR motif (M_(n+1)) on the C-terminusside, the amino acid locating upstream of the first amino acid of thenext PPR motif (M_(n+1)) by 2 positions, i.e., the -2nd amino acid; or

when any next PPR motif (M_(n+1)) does not exist on the C-terminus sideof the PPR motif (M_(n)), or 21 or more amino acids constituting anon-PPR motif exist between the PPR motif (M_(n)) and the next PPR motif(M_(n+1)) on the C-terminus side, the 2nd amino acid counted from theend (C-terminus side) of the amino acids constituting the PPR motif(M_(n)) is referred to as No. “ii” (-2) amino acid (No. “ii” (-2) A.A.),one PPR motif (M_(n)) contained in the protein is a PPR motif having aspecific combination of amino acids corresponding to a target DNA baseor target DNA base sequence as the three amino acids of No. 1 A.A., No.4 A.A., and No. “ii” (-2) A.A.

[2] The protein according to [1], wherein the combination of the threeamino acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. is acombination corresponding to a target DNA base or target DNA basesequence. In one embodiment, each PPR motif (M_(n)) contained in theDNA-binding protein is a PPR motif having a specific combination ofamino acids as the three amino acids of Number 1 AA, Number 4 AA, andNumber “ii” (-2) AA, such that the DNA-binding protein has at least 9combinations of the three amino acids corresponding to the respectivetarget DNA bases of the target DNA base sequence. Each combination ofthe three amino acids is determined according to any one of thefollowing definitions:(1-1) when No. 4 A.A. is glycine (G), No. 1 A.A. may be an arbitraryamino acid, and No. “ii” (-2) A.A. is aspartic acid (D), asparagine (N),or serine (S);(1-2) when No. 4 A.A. is isoleucine (I), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid;(1-3) when No. 4 A.A. is leucine (L), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid;(1-4) when No. 4 A.A. is methionine (M), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid;(1-5) when No. 4 A.A. is asparagine (N), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid;(1-6) when No. 4 A.A. is proline (P), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid;(1-7) when No. 4 A.A. is serine (S), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid;(1-8) when No. 4 A.A. is threonine (T), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid; and(1-9) when No. 4 A.A. is valine (V), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid.[3] The protein according to [1], wherein the combination of the threeamino acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. is acombination corresponding to a target DNA base or target DNA basesequence, and the combination of amino acids is determined according toany one of the following definitions:(2-1) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A. are an arbitrary amino acid, glycine, and aspartic acid,respectively, the PPR motif selectively binds to G;(2-2) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are glutamic acid, glycine, and aspartic acid, respectively,the PPR motif selectively binds to G;(2-3) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, glycine, and asparagine,respectively, the PPR motif selectively binds to A;(2-4) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are glutamic acid, glycine, and asparagine, respectively, thePPR motif selectively binds to A;(2-5) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, glycine, and serine,respectively, the PPR motif selectively binds to A, and next binds to C;(2-6) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, isoleucine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to T and C;(2-7) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, isoleucine, and asparagine,respectively, the PPR motif selectively binds to T, and next binds to C;(2-8) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, leucine, and an arbitrary aminoacid, respectively, the PPR motif selectively binds to T and C;(2-9) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, leucine, and aspartic acid,respectively, the PPR motif selectively binds to C;(2-10) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, leucine, and lysine,respectively, the PPR motif selectively binds to T;(2-11) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, methionine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to T;(2-12) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, methionine, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-13) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, methionine, and aspartic acid, respectively,the PPR motif selectively binds to T, and next binds to C;(2-14) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to C and T;(2-15) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-16) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, asparagine, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-17) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are glycine, asparagine, and aspartic acid, respectively, thePPR motif selectively binds to T;(2-18) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, asparagine, and aspartic acid, respectively,the PPR motif selectively binds to T;(2-19) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are threonine, asparagine, and aspartic acid, respectively,the PPR motif selectively binds to T;(2-20) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A. are valine, asparagine, and aspartic acid, respectively, thePPR motif selectively binds to T, and next binds to C;(2-21) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A. are tyrosine, asparagine, and aspartic acid, respectively, thePPR motif selectively binds to T, and next binds to C;(2-22) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and asparagine,respectively, the PPR motif selectively binds to C;(2-23) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, asparagine, and asparagine, respectively, thePPR motif selectively binds to C;(2-24) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are serine, asparagine, and asparagine, respectively, the PPRmotif selectively binds to C;(2-25) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, asparagine, and asparagine, respectively, the PPRmotif selectively binds to C;(2-26) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and serine,respectively, the PPR motif selectively binds to C;(2-27) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, asparagine, and serine, respectively, the PPRmotif selectively binds to C;(2-28) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and threonine,respectively, the PPR motif selectively binds to C;(2-29) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, asparagine, and threonine, respectively, the PPRmotif selectively binds to C;(2-30) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and tryptophan,respectively, the PPR motif selectively binds to C, and next binds to T;(2-31) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, asparagine, and tryptophan, respectively, thePPR motif selectively binds to T, and next binds to C;(2-32) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, proline, and an arbitrary aminoacid, respectively, the PPR motif selectively binds to T;(2-33) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, proline, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-34) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, proline, and aspartic acid, respectively,the PPR motif selectively binds to T;(2-35) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are tyrosine, proline, and aspartic acid, respectively, thePPR motif selectively binds to T;(2-36) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, serine, and an arbitrary aminoacid, respectively, the PPR motif selectively binds to A and G;(2-37) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, serine, and asparagine,respectively, the PPR motif selectively binds to A;(2-38) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, serine, and asparagine, respectively, thePPR motif selectively binds to A;(2-39) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, serine, and asparagine, respectively, the PPRmotif selectively binds to A;(2-40) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, threonine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to A and G;(2-41) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, threonine, and aspartic acid,respectively, the PPR motif selectively binds to G;(2-42) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, threonine, and aspartic acid, respectively, thePPR motif selectively binds to G;(2-43) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, threonine, and asparagine,respectively, the PPR motif selectively binds to A;(2-44) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, threonine, and asparagine, respectively,the PPR motif selectively binds to A;(2-45) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, threonine, and asparagine, respectively, thePPR motif selectively binds to A;(2-46) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, threonine, and asparagine, respectively, the PPRmotif selectively binds to A;(2-47) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, valine, and an arbitrary aminoacid, respectively, the PPR motif binds with A, C, and T, but does notbind to G;(2-48) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, valine, and aspartic acid, respectively, thePPR motif selectively binds to C, and next binds to A;(2-49) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, valine, and glycine,respectively, the PPR motif selectively binds to C; and(2-50) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, valine, and threonine,respectively, the PPR motif selectively binds to T.[4] The protein according to any one of [1] to [3], which contains 2 to30 of the PPR motifs (M_(n)) defined in [1].[5] The protein according to any one of [1] to [3], which contains 5 to25 of the PPR motifs (M_(n)) defined in [1].[6] The protein according to any one of [1] to [3], which contains 9 to15 of the PPR motifs (M_(n)) defined in [1].[7] The PPR protein according to [6], which consists of a sequenceselected from the amino acid sequence of SEQ ID NO: 1 containing 9 PPRmotifs, the amino acid sequence of SEQ ID NO: 2 containing 11 PPRmotifs, the amino acid sequence of SEQ ID NO: 3 containing 15 PPRmotifs, the amino acid sequence of SEQ ID NO: 4 containing 10 PPRmotifs, and the amino acid sequence of SEQ ID NO: 5 containing 11 PPRmotifs.[8] A method for identifying a DNA base or DNA base sequence that servesas a target of a DNA-binding protein containing one or more (preferably2 to 30) PPR motifs (M_(n)) defined in [1], wherein:

the DNA base or DNA base sequence is identified by determining presenceor absence of a DNA base corresponding to a combination of the threeamino acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. of the PPRmotif on the basis of any one of the definitions (1-1) to (1-9)mentioned in [2], and (2-1) to (2-50) mentioned in [3]. [9] A method foridentifying a PPR protein containing one or more (preferably 2 to 30)PPR motifs (M_(n)) defined in [1] that can bind to a target DNA base ortarget DNA having a specific base sequence, wherein:

the PPR protein is identified by determining presence or absence of acombination of the three amino acids of No. 1 A.A., No. 4 A.A., and No.“ii” (-2) A.A. corresponding to the target DNA base or a specific baseconstituting the target DNA on the basis of any one of the definitions(1-1) to (1-9) mentioned in [2], and (2-1) to (2-50) mentioned in [3].

[10] A method for controlling a function of DNA, which uses the proteinaccording to [1].[11] A complex consisting of a region comprising the protein accordingto [1], and a functional region bound together.[12] The complex according to [11], wherein the functional region isfused to the protein according to [1] on the C-terminus side of theprotein.[13] The complex according to [11] or [12], wherein the functionalregion is a DNA-cleaving enzyme, or a nuclease domain thereof, or atranscription control domain, and the complex functions as a targetsequence-specific DNA-cleaving enzyme or transcription control factor.[14] The complex according to [13], wherein the DNA-cleaving enzyme isthe nuclease domain of FokI (SEQ ID NO: 6).[15] A method for modifying a genetic substance of a cell comprising thefollowing steps:

preparing a cell containing a DNA having a target sequence; and

introducing the complex according to [11] into the cell so that theregion of the complex consisting of the protein binds to the DNA havinga target sequence, and therefore the functional region modifies the DNAhaving a target sequence.

[16] A method for identifying, recognizing, or targeting a DNA base orDNA having a specific base sequence by using a PPR protein containingone or more PPR motifs.[17] The method according to [16], wherein the protein contains one ormore PPR motifs in which three amino acids among the amino acidsconstituting the motif constitute a specific combination of amino acids.[18] The method according to [16] or [17], wherein the protein containsone or more PPR motifs (M_(n)) defined in [1].

Effect of the Invention

According to the present invention, a PPR motif that can binds to atarget DNA base, and a protein containing it can be provided. Byarranging two or more PPR motifs, a protein that can binds to a targetDNA having an arbitrary sequence or length can be provided.

According to the present invention, a target DNA of an arbitrary PPRprotein can be predicted and identified, and conversely, a PPR proteinthat binds to an arbitrary DNA can be predicted and identified.Prediction of such a target DNA sequence clarifies the genetic identitythereof, and increases possibility of use thereof. Furthermore,according to the present invention, functionalities of homologous genesof a gene of an industrially useful PPR protein showing amino acidpolymorphism at a high level can be determined on the basis ofdifference of the target DNA base sequences thereof.

Furthermore, according to the present invention, a novel DNA-cleavingenzyme using a PPR motif can also be provided. That is, by linking aprotein as a functional region with the PPR motif or PPR proteinprovided by the present invention, a complex containing a protein havinga binding activity for a specific nucleic acid sequence, and a proteinhaving a specific functionality can be prepared.

The functional region usable in the present invention is one that canimpart, among various functions, a function for any one of cleavage,transcription, replication, restoration, synthesis, modification, etc.of DNA. By choosing the sequence of the PPR motifs, which is thecharacteristic of the present invention, to determine a base sequence ofDNA as a target, almost all DNA sequences can be used as a target, andgenome edition using a function of the functional region such as thosefor cleavage, transcription, replication, restoration, synthesis,modification, etc. of DNA can be realized with such a target.

For example, when the functional region has a function for cleaving DNA,a complex comprising a PPR protein part prepared according to thepresent invention and a DNA-cleaving region linked together is provided.Such a complex can function as an artificial DNA-cleaving enzyme, whichrecognizes a base sequence of DNA as a target with the PPR protein part,and then cleaves DNA with the region for cleaving DNA. When thefunctional region has a transcription control function, a complexcomprising a PPR protein part prepared according to the presentinvention and a transcription control region for DNA linked together isprovided. Such a complex can function as an artificial transcriptioncontrol factor, which recognizes a base sequence of DNA as a target withthe PPR protein part, and then promotes transcription of the target DNA.

The present invention can further be utilized for a method fordelivering the aforementioned complex in a living body so that thecomplex functions in the living body, and preparation of transformantsutilizing a nucleic acid sequence (DNA and RNA) encoding a proteinobtained according to the present invention, as well as specificmodification, control, and impartation of a function in varioussituations in organisms (cells, tissues, and individuals).

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1C show conserved sequences and amino acid numbers of the PPRmotif. FIG. 1A shows the amino acids constituting the PPR motif definedin the present invention, and the amino acid numbers thereof (the aminoacid sequences P, S, L1, and L2 correspond to SEQ ID NO: 20, SEQ ID NO:21, SEQ ID NO: 22, and SEQ ID NO: 23, respectively). FIG. 1B showspositions of three amino acids (No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A.) that control binding base selectivity in the predictedstructure. FIG. 1C shows two examples of the structure of the PPR motif,and the positions of the amino acids on the predicted structure for eachcase. No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. are indicated withsticks of magenta color (dark gray in the case of monochratic display)in the conformational diagrams of the protein.

FIG. 2 summarizes the outlines of the structures of Arabidopsis thalianap63 (amino acid sequence of SEQ ID NO: 1), the GUN1 protein ofArabidopsis thaliana (amino acid sequence of SEQ ID NO: 2), pTac2 ofArabidopsis thaliana (amino acid sequence of SEQ ID NO: 3), DG1 (aminoacid sequences of SEQ ID NO: 4), and GRP23 of Arabidopsis thaliana(amino acid sequence of SEQ ID NO: 5), which are DNA-binding type PPRproteins that function in DNA metabolism, and the outline of the assaysystem for demonstrating that they bind to DNA.

FIG. 3 summarizes the amino acid frequencies of the amino acids at thethree positions bearing the nucleic acid recognition codes in the PPRmotif (No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A.) for the PPRmotifs of the PPR proteins (SEQ ID NOS: 1 to 5), for which DNA bindingproperty was suggested, and known RNA-binding type motifs.

FIG. 4-1 shows the positions of the PPR motifs included in the inside ofthe proteins, and the positions of the three amino acids bearing thenucleic acid recognition codes (No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A.) in the PPR motifs for each of (A) Arabidopsis thaliana p63(amino acid sequence of SEQ ID NO: 1) and (B) the GUN1 protein ofArabidopsis thaliana (amino acid sequence of SEQ ID NO: 2.

FIG. 4-2 shows the positions of the PPR motifs included in the inside ofthe proteins, and the positions of the three amino acids bearing thenucleic acid recognition codes (No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A.) in the PPR motifs for each of (C) pTac2 of Arabidopsisthaliana (amino acid sequence of SEQ ID NO: 3), and (D) DG1 (amino acidsequence of SEQ ID NO: 4).

FIG. 4-3 shows the positions of the PPR motifs included in the inside ofthe proteins, and the positions of the three amino acids bearing thenucleic acid recognition codes (No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A.) in the PPR motifs for (E) GRP23 of Arabidopsis thaliana(amino acid sequence of SEQ ID NO: 5).

FIG. 5 shows the evaluation of the sequence-specific DNA-bindingabilities of the PPR molecules. Artificial transcription factors wereprepared by fusing each of three kinds of DNA-binding type (regarded so)PPR molecules with VP64, which is a transcription activation domain, andwhether they could activate a luciferase reporter having each targetsequence was examined in a human cultured cell.

FIG. 6 shows comparison of the luciferase activities observed bycointroduction of pTac2-VP64 or GUN1-VP64 with pminCMV-luc2 as anegative control, or a reporter vector comprising 4 or 8 targetsequences. As a result, there was observed a tendency that the activityincreased with increase of the target sequence for the both molecules,and thus it was verified that these PPR-VP64 molecules specificallybound to each target sequence to function as a site-specifictranscription activator.

MODES FOR CARRYING OUT THE INVENTION

[PPR Motif and PPR Protein]

The “PPR motif” referred to in the present invention means a polypeptideconstituted with 30 to 38 amino acids and having an amino acid sequencethat shows, when the amino acid sequence is analyzed with a proteindomain search program on the web (for example, Pfam, Prosite, Uniprot,etc.), an E value not larger than a predetermined value (desirably E-03)obtained at PF01535 in the case of Pfam (http://pfam.sanger.ac.uk/), orPS51375 in the case of Prosite (http://www.expasy.org/prosite/), unlessotherwise indicated. The PPR motifs in various proteins are also definedin the Uniprot database (http://www.uniprot.org).

Although the amino acid sequence of the PPR motif is not highlyconserved in the PPR motif of the present invention, such a secondarystructure of helix, loop, helix, and loop as shown by the followingformula is conserved well.

[Formula 2]

(Helix A)-X-(Helix B)-L  (Formula 1)

The position numbers of the amino acids constituting the PPR motifdefined in the present invention are according to those defined in apaper of the inventors of the present invention (Kobayashi K, et al.,Nucleic Acids Res., 40, 2712-2723 (2012)). That is, the position numbersof the amino acids constituting the PPR motif defined in the presentinvention are substantially the same as the amino acid numbers definedfor PF01535 in Pfam, but correspond to numbers obtained by subtracting 2from the amino acid numbers defined for PS51375 in Prosite (for example,position 1 according to the present invention is position 3 of PS51375),and also correspond to numbers obtained by subtracting 2 from the aminoacid numbers of the PPR motif defined in Uniprot.

More precisely, in the present invention, the No. 1 amino acid is thefirst amino acid from which Helix A shown in the formula 1 starts. TheNo. 4 amino acid is the fourth amino acid counted from the No. 1 aminoacid. As for “ii” (-2)nd amino acid,

when a next PPR motif (M_(n+1)) contiguously exists on the C-terminusside of the PPR motif (M_(n)) (when there is no amino acid insertionbetween the PPR motifs, as in the cases of, for example, Motif Nos. 1,2, 3, 4, 6 and 7 in FIG. 4-1 (A)), the -2nd amino acid counted from theend (C-terminus side) of the amino acids constituting the PPR motif(M_(n)) is referred to as No. “ii” (-2) amino acid;

when a non-PPR motif (part that is not the PPR motif) consisting of 1 to20 amino acids exists between the PPR motif (M_(n)) and the next PPRmotif (M_(n+1)) on the C-terminus side (as in the cases of, for example,Motif Nos. 5 and 8 in FIG. 4-1 (A), and Motif Nos. 1, 2, 7 and 8 in FIG.4-3 (D)), the amino acid locating upstream of the first amino acid ofthe next PPR motif (M_(n+1)) by 2 positions, i.e., the −2nd amino acid,is referred to as No. “ii” (-2) amino acid (refer to FIG. 1); or

when any next PPR motif (M_(n+1)) does not exist on the C-terminus sideof the PPR motif (M_(n)) (as in the cases of, for example, Motif No. 9in FIG. 4-1 (A), and Motif No. 11 in FIG. 4-1 (B)), or 21 or more aminoacids constituting a non-PPR motif exist between the PPR motif (M_(n))and the next PPR motif (M_(n+1)) on the C-terminus side, the 2nd aminoacid counted from the end (C-terminus side) of the amino acidsconstituting the PPR motif (M_(n)) is referred to as No. “ii” (-2) aminoacid.

The “PPR protein” referred to in the present invention means a PPRprotein having two or more of the aforementioned PPR motifs, unlessotherwise indicated. The term “protein” used in this specification meansany substance consisting of a polypeptide (chain consisting of two ormore amino acids bound through peptide bonds), and also includes thoseconsisting of a comparatively low molecular weight polypeptide, unlessotherwise indicated. The “amino acid” referred to in the presentinvention means a usual amino acid molecule, as well as an amino acidresidue constituting a peptide chain. Which the term means will beapparent to those skilled in the art from the context.

Many PPR proteins exist in plants, and 500 proteins and about 5000motifs can be found in Arabidopsis thaliana. PPR motifs and PPR proteinsof various amino acid sequences also exist in many land plants such asrice, poplar, and selaginella. It is known that some PPR proteins areimportant factors for obtaining F1 seeds for hybrid vigor as fertilityrestoration factors that are involved in formation of pollen (malegamete). It has been clarified that some PPR proteins are involved inspeciation, similarly in fertility restoration. It has also beenclarified that almost all the PPR proteins act on RNA in mitochondria orchloroplasts.

It is known that, in animals, anomaly of the PPR protein identified asLRPPRC causes Leigh syndrom French Canadian (LSFC, Leigh's syndrome,subacute necrotizing encephalomyelopathy).

The term “selective” used for a property of a PPR motif for binding witha DNA base in the present invention means that a binding activity forany one base among the DNA bases is higher than binding activities forthe other bases, unless otherwise indicates. Those skilled in the artcan confirm this selectivity by planning an experiment, or it can alsobe obtained by calculation as described in the examples mentioned inthis specification.

The DNA base referred to in the present invention means a base ofdeoxyribonucleotide constituting DNA, and specifically, it means any ofadenine (A), guanine (G), cytosine (C), and thymine (T), unlessotherwise indicated. Although the PPR protein may have selectivity to abase in DNA, it does not bind to a nucleic acid monomer.

Although search methods for conserved amino acid sequence as the PPRmotif had been established before the present invention wasaccomplished, any rule concerning selective binding with DNA base hadnot been discovered at all.

Findings Provided by the Present Invention

The following findings are provided by the present invention.

(I) Information about Positions of Amino Acids Important for SelectiveBinding

Specifically, under the following definitions:

the first amino acid of Helix A of the PPR motif is referred to as No. 1amino acid (No. 1 A.A.), the fourth amino acid as No. 4 amino acid (No.4 A.A.), and

when a next PPR motif (M_(n+1)) contiguously exists on the C-terminusside of the PPR motif (M_(n)) (when there is no amino acid insertionbetween the PPR motifs), the -2nd amino acid counted from the end(C-terminus side) of the amino acids constituting the PPR motif (M_(n));

when a non-PPR motif consisting of 1 to 20 amino acids exist between thePPR motif (M_(n)) and the next PPR motif (M_(n+1)) on the C-terminusside, the amino acid locating upstream of the first amino acid of thenext PPR motif (M_(n+1)) by 2 positions, i.e., the -2nd amino acid; or

when any next PPR motif (M_(n+1)) does not exist on the C-terminus sideof the PPR motif (M_(n)), or 21 or more amino acids constituting anon-PPR motif exist between the PPR motif (M_(n)) and the next PPR motif(M_(n+1)) on the C-terminus side, the 2nd amino acid counted from theend (C-terminus side) of the amino acids constituting the PPR motif(M_(n))

is referred to as No. “ii” (-2) amino acid (No. “ii” (-2) A.A.),combination of the three amino acids, the first and fourth amino acidsof the helix (Helix A), No. 1 and No. 4 amino acids, and No. “ii” (-2)A.A. defined above (No. 1 A.A., No. 4 A.A. and No. “ii” (-2) A.A.) isimportant for selective binding to a DNA base, and to what kind of DNAbase the motif binds can be determined on the basis of the combination.

The present invention is based on the findings concerning thecombination of the three amino acids, No. 1 A.A., No. 4 A.A., and No.“ii” (-2) A.A., found by the inventors of the present invention.Specifically:

(1-1) when No. 4 A.A. is glycine (G), No. 1 A.A. may be an arbitraryamino acid, No. “ii” (-2) A.A. is aspartic acid (D), asparagine (N), orserine (S), and the combination of No. 1 A.A., and No. “ii” (-2) A.A.may be, for example:

a combination of an arbitrary amino acid and aspartic acid (D) (*GD),

preferably a combination of glutamic acid (E) and aspartic acid (D)(EGD),

a combination of an arbitrary amino acid and asparagine (N) (*GN),

preferably a combination of glutamic acid (E) and asparagine (N) (EGN),or

a combination of an arbitrary amino acid and serine (S) (*GS);

(1-2) when No. 4 A.A. is isoleucine (I), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and asparagine (N) (*IN);

(1-3) when No. 4 A.A. is leucine (L), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and aspartic acid (D) (*LD), or

a combination of an arbitrary amino acid and lysine (K) (*LK);

(1-4) when No. 4 A.A. is methionine (M), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and aspartic acid (D) (*MD), or

a combination of isoleucine (I) and aspartic acid (D) (IMD);

(1-5) when No. 4 A.A. is asparagine (N), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and aspartic acid (D) (*ND),

a combination of any one of phenylalanine (F), glycine (G), isoleucine(I), threonine (T), valine (V) and tyrosines (Y), and aspartic acid (D)(FND, GND, IND, TND, VND, or YND),

a combination of an arbitrary amino acid and asparagine (N) (*NN),

a combination of any one of isoleucine (I), serine (S) and valine (V),and asparagine (N) (INN, SNN or VNN)

a combination of an arbitrary amino acid and serine (S) (*NS),

a combination of valine (V) and serine (S) (VNS),

a combination of an arbitrary amino acid and threonine (T) (*NT),

a combination of valine (V) and threonine (T) (VNT),

a combination of an arbitrary amino acid and tryptophan (W) (*NW), or

a combination of isoleucine (I) and tryptophan (W) (INW);

(1-6) when No. 4 A.A. is proline (P), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and aspartic acid (D) (*PD),

a combination of phenylalanine (F) and aspartic acid (D) (FPD), or

a combination of tyrosine (Y) and aspartic acid (D) (YPD);

(1-7) when No. 4 A.A. is serine (S), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and asparagine (N) (*SN),

a combination of phenylalanine (F) and asparagine (N) (FSN), or

a combination of valine (V) and asparagine (N) (VSN);

(1-8) when No. 4 A.A. is threonine (T), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of an arbitrary amino acid and aspartic acid (D) (*TD),

a combination of valine (V) and aspartic acid (D) (VTD),

a combination of an arbitrary amino acid and asparagine (N) (*TN),

a combination of phenylalanine (F) and asparagine (N) (FTN),

a combination of isoleucine (I) and asparagine (N) (ITN), or

a combination of valine (V) and asparagine (N) (VTN); and

(1-9) when No. 4 A.A. is valine (V), each of No. 1 A.A. and No. “ii”(-2) A.A. may be an arbitrary amino acid, and the combination of No. 1A.A., and No. “ii” (-2) A.A. may be, for example:

a combination of isoleucine (I) and aspartic acid (D) (IVD),

a combination of an arbitrary amino acid and glycine (G) (*VG), or

a combination of an arbitrary amino acid and threonine (T) (*VT).

(II) Information about Correspondence of Combination of Three AminoAcids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A., and DNA Base

The protein is a protein determined on the basis of, specifically, thefollowing definitions, and having a selective DNA base-binding property:

(2-1) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, glycine, and aspartic acid,respectively, the PPR motif selectively binds to G;(2-2) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are glutamic acid, glycine, and aspartic acid, respectively,the PPR motif selectively binds to G;(2-3) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, glycine, and asparagine,respectively, the PPR motif selectively binds to A;(2-4) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are glutamic acid, glycine, and asparagine, respectively, thePPR motif selectively binds to A;(2-5) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, glycine, and serine,respectively, the PPR motif selectively binds to A, and next binds to C;(2-6) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, isoleucine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to T and C;(2-7) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, isoleucine, and asparagine,respectively, the PPR motif selectively binds to T, and next binds to C;(2-8) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, leucine, and an arbitrary aminoacid, respectively, the PPR motif selectively binds to T and C;(2-9) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, leucine, and aspartic acid,respectively, the PPR motif selectively binds to C;(2-10) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, leucine, and lysine,respectively, the PPR motif selectively binds to T;(2-11) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, methionine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to T;(2-12) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, methionine, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-13) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, methionine, and aspartic acid, respectively,the PPR motif selectively binds to T, and next binds to C;(2-14) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to C and T;(2-15) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-16) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, asparagine, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-17) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are glycine, asparagine, and aspartic acid, respectively, thePPR motif selectively binds to T;(2-18) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, asparagine, and aspartic acid, respectively,the PPR motif selectively binds to T;(2-19) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are threonine, asparagine, and aspartic acid, respectively,the PPR motif selectively binds to T;(2-20) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A. are valine, asparagine, and aspartic acid, respectively, thePPR motif selectively binds to T, and next binds to C;(2-21) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A. are tyrosine, asparagine, and aspartic acid, respectively, thePPR motif selectively binds to T, and next binds to C;(2-22) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and asparagine,respectively, the PPR motif selectively binds to C;(2-23) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, asparagine, and asparagine, respectively, thePPR motif selectively binds to C;(2-24) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are serine, asparagine, and asparagine, respectively, the PPRmotif selectively binds to C;(2-25) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, asparagine, and asparagine, respectively, the PPRmotif selectively binds to C;(2-26) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and serine,respectively, the PPR motif selectively binds to C;(2-27) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, asparagine, and serine, respectively, the PPRmotif selectively binds to C;(2-28) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and threonine,respectively, the PPR motif selectively binds to C;(2-29) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, asparagine, and threonine, respectively, the PPRmotif selectively binds to C;(2-30) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, asparagine, and tryptophan,respectively, the PPR motif selectively binds to C, and next binds to T;(2-31) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, asparagine, and tryptophan, respectively, thePPR motif selectively binds to T, and next binds to C;(2-32) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, proline, and an arbitrary aminoacid, respectively, the PPR motif selectively binds to T;(2-33) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, proline, and aspartic acid,respectively, the PPR motif selectively binds to T;(2-34) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, proline, and aspartic acid, respectively,the PPR motif selectively binds to T;(2-35) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are tyrosine, proline, and aspartic acid, respectively, thePPR motif selectively binds to T;(2-36) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, serine, and an arbitrary aminoacid, respectively, the PPR motif selectively binds to A and G;(2-37) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, serine, and asparagine,respectively, the PPR motif selectively binds to A;(2-38) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, serine, and asparagine, respectively, thePPR motif selectively binds to A;(2-39) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, serine, and asparagine, respectively, the PPRmotif selectively binds to A;(2-40) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, threonine, and an arbitraryamino acid, respectively, the PPR motif selectively binds to A and G;(2-41) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, threonine, and aspartic acid,respectively, the PPR motif selectively binds to G;(2-42) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, threonine, and aspartic acid, respectively, thePPR motif selectively binds to G;(2-43) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, threonine, and asparagine,respectively, the PPR motif selectively binds to A;(2-44) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are phenylalanine, threonine, and asparagine, respectively,the PPR motif selectively binds to A;(2-45) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, threonine, and asparagine, respectively, thePPR motif selectively binds to A;(2-46) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are valine, threonine, and asparagine, respectively, the PPRmotif selectively binds to A;(2-47) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, valine, and an arbitrary aminoacid, respectively, the PPR motif binds with A, C, and T, but does notbind to G;(2-48) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are isoleucine, valine, and aspartic acid, respectively, thePPR motif selectively binds to C, and next binds to A;(2-49) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, valine, and glycine,respectively, the PPR motif selectively binds to C; and(2-50) when the three amino acids, No. 1 A.A., No. 4 A.A., and No. “ii”(-2) A.A., are an arbitrary amino acid, valine, and threonine,respectively, the PPR motif selectively binds to T.

Combination of amino acids of specific positions and binding propertywith a DNA base can be confirmed by experiments. Experiments for suchpurposes include preparation of a PPR motif or a protein containing twoor more PPR motifs, preparation of a substrate DNA, and binding propertytest (for example, gel shift assay). These experiments are well known tothose skilled in the art, and as for more specific procedures andconditions, for example, Patent document 2 can be referred to.

[Use of PPR Motif and PPR Protein]

Identification and Design

One PPR motif recognizes a specific one kind of base of DNA, and two ormore contiguous PPR motifs can recognize continuous bases in a DNAsequence. Further, according to the present invention, by appropriatelychoosing amino acids at specific positions, PPR motifs selective foreach of A, T, and C can be chosen or designed, and a protein containingan appropriate continuation of such PPR motifs can recognize acorresponding specific sequence. Therefore, according to the presentinvention, a naturally occurring PPR protein that selectively binds toDNA having a specific base sequence can be predicted or identified, orconversely, DNA as a target of binding of a PPR protein can be predictedand identified. Prediction or identification of such a target is usefulfor clarifying genetic identity of the target, and is also useful from aviewpoint that such prediction or identification may expandapplicability of the target.

Furthermore, according to the present invention, a PPR motif that canselectively bind to a desired DNA base, and a protein having two or morePPR motifs that can bind to a desired DNA in a sequence-specific mannercan be designed. In such design, as for the part other than the aminoacids at the important positions in the PPR motif, sequence informationon PPR motifs of naturally occurring type in DNA-binding type PPRproteins such as those of SEQ ID NOS: 1 to 5 can be referred to.Further, the motif or protein may also be designed by using a motif orprotein of naturally occurring type as a whole, and replacing only theamino acids of the corresponding positions. Although the number ofrepetitions of PPR motifs can be appropriately chosen according to atarget sequence, it may be, for example, 2 or more, preferably 2 to 30,more preferably 5 to 25, most preferably 9 to 15.

In the designing, amino acids other than those of the combination of theamino acids of No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A. may betaken into consideration. For example, selection of the amino acids ofNo. 8 and No. 12 described in Patent document 2 mentioned above may beimportant for exhibiting a DNA-binding activity. According to theresearches of the inventors of the present invention, the No. 8 aminoacid of a certain PPR motif and the No. 12 amino acid of the same PPRmotif may cooperate in binding with DNA. The No. 8 amino acid may be abasic amino acid, preferably lysine, or an acidic amino acid, preferablyaspartic acid, and the No. 12 amino acid may be a basic amino acid,neutral amino acid, or hydrophobic amino acid.

A designed motif or protein can be prepared by methods well known tothose skilled in the art. That is, the present invention provides a PPRmotif that selectively binds to a specific DNA base, and a PPR proteinthat specifically binds to DNA having a specific sequence, in whichattention is paid to the combination of the amino acids of No. 1 A.A.,No. 4 A.A., and No. “ii” (-2) A.A. Such a motif and protein can beprepared even in a comparatively large amount by methods well known tothose skilled in the art, and such methods may comprise determining anucleic acid sequence encoding a target motif or protein from the aminoacid sequence of the target motif or protein, cloning it, and preparinga transformant that produces the target motif or protein.

Preparation of Complex and Use Thereof

The PPR motif or PPR protein provided by the present invention can bemade into a complex by binding a functional region. The functionalregion generally refers to a part having such a function as a specificbiological function exerted in a living body or cell, for example,enzymatic function, catalytic function, inhibitory function, promotionfunction, etc, or a function as a marker. Such a region consists of, forexample, a protein, peptide, nucleic acid, physiologically activesubstance, or drug.

According to the present invention, by binding a functional region tothe PPR protein, the target DNA sequence-binding function exerted by thePPR protein, and the function exerted by the functional region can beexhibited in combination. For example, if a protein having aDNA-cleaving function (for example, restriction enzyme such as FokI) ora nuclease domain thereof is used as the functional region, the complexcan function as an artificial DNA-cleaving enzyme.

In order to produce such a complex, methods generally available in thistechnical field can be used, and there are known a method ofsynthesizing such a complex as one protein molecule, a method ofseparately synthesizing two or more members of proteins, and thencombining them to form a complex, and so forth.

In the case of the method of synthesizing a complex as one proteinmolecule, for example, a protein complex can be designed so as tocomprise a PPR protein and a cleaving enzyme bound to the C-terminus ofthe PPR protein via an amino acid linker, an expression vector structurefor expressing the protein complex can be constructed, and the targetcomplex can be expressed from the structure. As such a preparationmethod, the method described in Japanese Patent Application No.2011-242250, and so forth can be used.

For binding the PPR protein and the functional region protein, anybinding means known in this technical field may be used, includingbinding via an amino acid linker, binding utilizing specific affinitysuch as binding between avidin and biotin, binding utilizing anotherchemical linker, and so forth.

The functional region usable in the present invention refers to a regionthat can impart any one of various functions such as those for cleavage,transcription, replication, restoration, synthesis, or modification ofDNA, and so forth. By choosing the sequence of the PPR motif to define aDNA base sequence as a target, which is the characteristic of thepresent invention, substantially any DNA sequence may be used as thetarget, and with such a target, genome edition utilizing the function ofthe functional region such as those for cleavage, transcription,replication, restoration, synthesis, or modification of DNA can berealized.

For example, when the function of the functional region is a DNAcleavage function, there is provided a complex comprising a PPR proteinpart prepared according to the present invention and a DNA cleavageregion bound together. Such a complex can function as an artificialDNA-cleaving enzyme that recognizes a base sequence of DNA as a targetby the PPR protein part, and then cleaves DNA by the DNA cleavageregion.

An example of the functional region having a cleavage function usablefor the present invention is a deoxyribonuclease (DNase), whichfunctions as an endodeoxyribonuclease. As such a DNase, for example,endodeoxyribonucleases such as DNase A (e.g., bovine pancreaticribonuclease A, PDB 2AAS), DNase H and DNase I, restriction enzymesderived from various bacteria (for example, FokI (SEQ ID NO: 6) etc.)and nuclease domains thereof can be used. Such a complex comprising aPPR protein and a functional region does not exist in the nature, and isnovel.

When the function of the functional region is a transcription controlfunction, there is provided a complex comprising a PPR protein partprepared according to the present invention and a DNA transcriptioncontrol region bound together. Such a complex can function as anartificial transcription control factor, which recognizes a basesequence of DNA as a target by the PPR protein part, and then controlstranscription of the target DNA.

The functional region having a transcription control function usable forthe present invention may be a domain that activates transcription, ormay be a domain that suppresses transcription. Examples of thetranscription control domain include VP16, VP64, TA2, STAT-6, and p65.Such a complex comprising a PPR protein and a transcription controldomain does not exist in the nature, and is novel.

Further, the complex obtainable according to the present invention maydeliver a functional region in a living body or cell in a DNAsequence-specific manner, and allow it to function. It thereby makes itpossible to perform modification or disruption in a DNAsequence-specific manner in a living body or cell, like proteincomplexes utilizing a zinc finger protein (Non-patent documents 1 and 2mentioned above) or TAL effecter (Non-patent document 3 and Patentdocument 1 mentioned above), and thus it becomes possible to impart anovel function, i.e., function for cleavage of DNA and genome editionutilizing that function. Specifically, with a PPR protein comprising twoor more PPR motifs that can bind with a specific base linked together, aspecific DNA sequence can be recognized. Then, genome edition of therecognized DNA region can be realized by the functional region bound tothe PPR protein using the function of the functional region.

Furthermore, by binding a drug to the PPR protein that binds to a DNAsequence in a DNA sequence-specific manner, the drug may be delivered tothe neighborhood of the DNA sequence as the target. Therefore, thepresent invention provides a method for DNA sequence-specific deliveryof a functional substance.

It has been clarified that the PPR protein used as a material in thepresent invention works to specify an edition position for DNA edition,and such a PPR motif having specific amino acids arranged at thepositions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2)A.A. recognizes a specific base on DNA, and then exhibits theDNA-binding activity thereof. On the basis of such a characteristic, aPPR protein of this type that has specific amino acids arranged at thepositions of the residues of No. 1 A.A., No. 4 A.A., and No. “ii” (-2)A.A. can be expected to recognize a base on DNA specific to each PPRprotein, and as a result, introduce base polymorphism, or to be used ina treatment of a disease or condition resulting from a basepolymorphism, and in addition, it is considered that the combination ofsuch a PPR protein with such another functional region as mentionedabove contribute to modification or improvement of functions forrealizing cleavage of DNA for genome edition.

Moreover, an exogenous DNA-cleaving enzyme can be fused to theC-terminus of the PPR protein. Alternatively, by improving binding DNAbase selectivity of the PPR motif on the N-terminus side, a DNAsequence-specific DNA-cleaving enzyme can also be constituted. Moreover,such a complex to which a marker part such as GFP is bound can also beused for visualization of a desired DNA in vivo.

EXAMPLES Example 1: Collection of PPR Proteins and Target SequencesThereof Used for DNA Edition

By referring to the information provided in the prior art references(Non-patent documents 11 to 15), structures and functions of the p63protein (SEQ ID NO: 1), GUN1 protein (SEQ ID NO: 2), pTac2 protein (SEQID NO: 3), DG1 protein (SEQ ID NO: 4), and GRP23 protein (SEQ ID NO: 5)were analyzed.

To the PPR motif structures in such proteins, amino acid numbers definedin the present invention were imparted together with the information ofthe Uniprot database (http://www.uniprot.org/). The PPR motifs containedin the five kinds of PPR proteins of Arabidopsis thaliana (SEQ ID NOS: 1to 5) used for the experiment, and the amino acid numbers thereof areshown in FIG. 3.

Specifically, amino acid frequencies for the amino acids at the threepositions (No. 1 A.A., No. 4 A.A., and No. “ii” (-2) A.A.) responsiblefor the nucleic acid recognition codes in the PPR motifs considered tobe important at the time of targeting RNA in the aforementioned p63protein (SEQ ID NO: 1), GUN1 protein (SEQ ID NO: 2), pTac2 protein (SEQID NO: 3), DG1 protein (SEQ ID NO: 4), and GRP23 protein (SEQ ID NO: 5)were compared with those of RNA-binding type motifs.

The p63 protein of Arabidopsis thaliana (SEQ ID NO: 1) has 9 PPR motifs,and the positions of the residues of No. 1 A.A., No. 4 A.A., and No.“ii” (-2) A.A. in the amino acid sequence are as summarized in thefollowing table and FIG. 3.

TABLE 1 Code Base to be bound (ratio) A₁ A₄ L_(ii) (1, 4, ii) A C G TPPR 230, V 233, R 263, S *R* 0.25 0.07 0.06 0.62 motif 1 PPR 265, F 268,D 297, S *D* 0.25 0.24 0.23 0.29 motif 2 PPR 299, L 302, K 322, D *KD0.20 0.18 0.28 0.34 motif 3 PPR 334, Q 377, A 367, N *AN 0.45 0.18 0.050.32 motif 4 PPR 369, R 372, K 399, Y *K* 0.17 0.32 0.23 0.29 motif 5PPR 401, E 404, L 434, S *LS 0.22 0.37 0.06 0.34 motif 6 PPR 436, S 439,S 469, E *SE 0.58 0.07 0.10 0.25 motif 7 PPR 471, T 474, D 505, M *D*0.25 0.24 0.23 0.29 motif 8 PPR 507, N 510, M 540, R *M* 0.13 0.14 0.220.51 motif 9

The GUN1 protein of Arabidopsis thaliana (SEQ ID NO: 2) has 11 PPRmotifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., andNo. “ii” (-2) A.A. in the amino acid sequence are as summarized in thefollowing table and FIG. 3.

TABLE 2 Code Base to be bound (ratio) A₁ A₄ L_(ii) (1, 4, ii) A C G TPPR 234, K 237, S 267, T *S* 0.41 0.12 0.22 0.25 motif 1 PPR 269, Y 272,S 302, N *SN 0.62 0.07 0.04 0.26 motif 2 PPR 304, V 307, N 338, D VND0.06 0.21 0.06 0.66 motif 3 PPR 340, I 343, N 373, D IND 0.14 0.24 0.120.50 motif 4 PPR 375, F 378, N 408, N FNN 0.24 0.21 0.24 0.31 motif 5PPR 410, V 413, S 443, D VSD 0.33 0.24 0.23 0.20 motif 6 PPR 445, V 448,N 478, D VND 0.06 0.21 0.06 0.66 motif 7 PPR 480, V 483, N 513, N VNN0.17 0.48 0.09 0.26 motif 8 PPR 515, L 518, S 548, D *SD 0.20 0.17 0.390.24 motif 9 PPR 550, V 553, S 583, N VSN 0.57 0.09 0.05 0.30 motif 10PPR 585, V 588, N 620, A *N* 0.10 0.33 0.10 0.48 motif 11

The pTac2 protein of Arabidopsis thaliana (SEQ ID NO: 3) has 15 PPRmotifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., andNo. “ii” (-2) A.A. in the amino acid sequence are as summarized in thefollowing table and FIG. 3.

TABLE 3 Code Base to be bound A₁ A₄ L_(ii) (1, 4, ii) A C G T PPR 106, N109, A 140, N *AN 0.45 0.18 0.05 0.32 motif 1 PPR 142, H 145, T 175, S*TS 0.37 0.29 0.15 0.19 motif 2 PPR 177, F 180, T 210, S *TS 0.37 0.290.15 0.19 motif 3 PPR 212, L 215, N 246, D LND 0.08 0.15 0.23 0.54 motif4 PPR 248, V 251, N 281, D VND 0.06 0.21 0.06 0.66 motif 5 PPR 283, T286, S 316, D TSD 0.14 0.18 0.14 0.54 motif 6 PPR 318, T 321, N 351, NTNN 0.08 0.49 0.17 0.26 motif 7 PPR 353, N 356, S 386, D *SD 0.20 0.170.39 0.24 motif 8 PPR 388, A 491, N 421, D AND 0.07 0.05 0.14 0.74 motif9 PPR 423, E 426, E 456, S B.G. 0.25 0.21 0.18 0.36 motif 10 PPR 458, K461, T 491, S *TS 0.37 0.29 0.15 0.19 motif 11 PPR 493, E 496, H 526, N*H* 0.17 0.34 0.06 0.43 motif 12 PPR 528, D 531, N 561, D *ND 0.11 0.170.10 0.62 motif 13 PPR 563, R 566, E 596, S B.G. 0.25 0.21 0.18 0.36motif 14 PPR 598, M 601, C 631. I *C* 0.55 0.10 0.21 0.14 motif 15 (B.G.means background)

The DG1 protein of Arabidopsis thaliana (SEQ ID NO: 4) has 10 PPRmotifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., andNo. “ii” (-2) A.A. in the amino acid sequence are as summarized in thefollowing table and FIG. 3.

TABLE 4 Code Base to be bound A₁ A₄ L_(ii) (1, 4, ii) A C G T PPR 256, F259, T 290, D *TD 0.10 0.10 0.67 0.13 motif 1 PPR 292, A 295, H 340, D*H* 0.17 0.34 0.06 0.43 motif 2 PPR 342, V 345, N 375, N VNN 0.17 0.480.09 0.26 motif 3 PPR 377, A 380, G 410, K *G* 0.29 0.13 0.31 0.27 motif4 PPR 412, I 415, K 445, T *K* 0.17 0.32 0.23 0.29 motif 5 PPR 477, S450, Y 481, L B.G. 0.25 0.21 0.18 0.36 motif 6 PPR 483, I 486, T 515, NITN 0.79 0.06 0.05 0.10 motif 7 PPR 517, G 520, N 553, N *NN 0.12 0.440.13 0.30 motif 8 PPR 555, Y 558, S 588, D YSD 0.25 0.15 0.39 0.20 motif9 PPR 590, T 593, A 623, H *AH 0.41 0.08 0.07 0.45 motif 10 (B.G. meansbackground)

The GRP23 protein of Arabidopsis thaliana (SEQ ID NO: 5) has 11 PPRmotifs, and the positions of the residues of No. 1 A.A., No. 4 A.A., andNo. “ii” (-2) A.A. in the amino acid sequence are as summarized in thefollowing table and FIG. 3.

TABLE 5 Code Base to be bound A₁ A₄ L_(ii) (1, 4, ii) A C G T PPR 181, F184, N 215, N FNN 0.24 0.21 0.24 0.31 motif 1 PPR 217, V 220, N 251, SVNS 0.07 0.61 0.05 0.27 motif 2 PPR 253, V 256, R 286, D *RD 0.25 0.070.06 0.62 motif 3 PPR 288, T 291, N 321, D TND 0.14 0.08 0.07 0.71 motif4 PPR 323, I 326, A 356, H *AH 0.41 0.08 0.07 0.45 motif 5 PPR 358, P361, N 396, N *NN 0.12 0.44 0.13 0.30 motif 6 PPR 398, D 401, G 435, D*GD 0.09 0.09 0.59 0.25 motif 7 PPR 437, L 404, C 470, D *CD 0.30 0.150.35 0.20 motif 8 PPR 472, P 475, R 505, V *R* 0.25 0.07 0.06 0.62 motif9 PPR 507, D 510, A 540, D *AD 0.10 0.22 0.39 0.29 motif 10 PPR 542, S545, D 575, T *D* 0.25 0.24 0.23 0.29 motif 11 (B.G. means background)

The amino acid frequencies for these positions were confirmed for eachprotein, and compared with the amino acid frequencies for the samepositions of the RNA-binding type motifs. The results are shown in FIG.2. It became clear that the tendencies of the amino acid frequencies inthe PPR motifs of the PPR proteins for which DNA-binding property issuggested, and the RNA-binding type motifs substantially agreed witheach other. That is, it became clear that the PPR proteins that act tobind to DNA bind with nucleic acids according to same sequence rules asthose of the PPR proteins that act to bind to RNA, and the RNArecognition codes described in the pending patent application of theinventors of the present invention (PCT/JP2012/077274) can be applied asthe DNA recognition codes of the PPR proteins that act to bind to DNA.

With reference to the RNA recognition codes described in the non-patentdocument (Yagi, Y. et al., Plos One, 2013, 8, e57286), the DNA-bindingtype PPR motifs that selectively bind to each corresponding base wereevaluated. More precisely, a chi square test was performed on the basisof occurrence nucleotide frequencies shown in Table 6 and expectednucleotide frequencies calculated from the background frequencies. Thetest was performed for each base (NT), purine or pyrimidine (AG or CT,PY), hydrogen bond group (AT or GC, HB), or amino or keto form (AC orGT). Significant value was defined as P<0.06 (5E−02, 5% significancelevel), and when a significant value was obtained in any of the tests,the combination of No. 1 amino acid, No. 4 amino acid, and No. “ii” (-2)amino acid was chosen.

TABLE 6 Base selectivity of DNA-binding code NSRs occurrence of theProbabilitiy matrix Subtraction for background (1, 4, ii) NSR(s) A C G TA C G T *GD 14 0.10 0.06 0.57 0.28 −0.16 −0.15 0.40 −0.08 EGD 8 0.070.05 0.69 0.19 −0.19 −0.16 0.52 −0.17 *GN 11 0.55 0.10 0.04 0.31 0.29−0.11 −0.13 −0.05 EGN 5 0.63 0.06 0.05 0.25 0.37 −0.15 −0.12 −0.11 *GS 30.57 0.23 0.06 0.14 0.31 0.02 −0.11 −0.22 *I* 15 0.15 0.29 0.10 0.45−0.11 0.08 −0.07 0.09 *IN 4 0.17 0.28 0.06 0.50 −0.09 0.07 −0.11 0.14*L* 23 0.20 0.30 0.03 0.47 −0.06 0.09 −0.14 0.11 *LD 5 0.19 0.47 0.050.28 −0.07 0.26 −0.12 −0.08 *LK 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.110.41 *M* 10 0.14 0.15 0.15 0.56 −0.12 −0.06 −0.02 0.20 *MD 9 0.15 0.130.17 0.55 −0.11 −0.08 0.00 0.19 IMD 4 0.09 0.24 0.06 0.62 −0.17 0.03−0.11 0.26 *N* 147 0.11 0.33 0.10 0.45 −0.15 0.12 −0.07 0.09 ND 72 0.110.18 0.10 0.61 −0.15 −0.03 −0.07 0.25 FND 13 0.23 0.19 0.10 0.49 −0.03−0.02 −0.07 0.13 GND 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.11 0.41 IND 50.22 0.13 0.05 0.60 −0.04 −0.08 −0.12 0.24 TND 3 0.15 0.08 0.06 0.72−0.11 −0.13 −0.11 0.36 VND 23 0.06 0.25 0.06 0.63 −0.20 0.04 −0.11 0.27YND 6 0.08 0.30 0.11 0.52 −0.18 0.09 −0.06 0.16 *NN 34 0.15 0.45 0.140.27 −0.11 0.24 −0.03 −0.09 INN 7 0.12 0.49 0.05 0.34 −0.14 0.28 −0.12−0.02 SNN 3 0.09 0.60 0.06 0.24 −0.17 0.39 −0.11 −0.12 VNN 10 0.20 0.530.04 0.23 −0.06 0.32 −0.13 −0.13 *NS 13 0.11 0.47 0.07 0.36 −0.15 0.26−0.10 0.00 VNS 5 0.08 0.66 0.05 0.21 −0.18 0.45 −0.12 −0.15 *NT 13 0.120.52 0.13 0.24 −0.14 0.31 −0.04 −0.12 VNT 5 0.08 0.57 0.05 0.30 −0.180.36 −0.12 −0.06 *NW 11 0.14 0.32 0.13 0.41 −0.12 0.11 −0.04 0.05 INW 30.09 0.29 0.06 0.56 −0.17 0.08 −0.11 0.20 *P* 17 0.10 0.06 0.11 0.73−0.16 −0.15 −0.06 0.37 *PD 9 0.06 0.09 0.10 0.75 −0.20 −0.12 −0.07 0.39FPD 3 0.09 0.08 0.06 0.77 −0.17 −0.13 −0.11 0.41 YPD 3 0.09 0.08 0.060.77 −0.17 −0.13 −0.11 0.41 *S* 49 0.38 0.13 0.20 0.29 0.12 −0.08 0.03−0.07 *SN 18 0.63 0.08 0.05 0.24 0.37 −0.13 −0.12 −0.12 FSN 7 0.63 0.130.08 0.16 0.37 −0.08 −0.09 −0.20 VSN 6 0.60 0.10 0.05 0.25 0.34 −0.11−0.12 −0.11 *T* 86 0.45 0.09 0.31 0.15 0.19 −0.12 0.14 −0.21 *TD 32 0.130.12 0.61 0.14 −0.13 −0.09 0.44 −0.22 VTD 7 0.07 0.06 0.67 0.20 −0.19−0.15 0.50 −0.16 *TN 31 0.66 0.08 0.13 0.13 0.40 −0.13 −0.04 −0.23 FTN 40.75 0.07 0.06 0.12 0.49 −0.14 −0.11 −0.24 ITN 5 0.77 0.06 0.05 0.110.51 −0.15 −0.12 −0.25 VTN 10 0.63 0.13 0.15 0.09 0.37 −0.08 −0.02 −0.27*V* 48 0.29 0.21 0.08 0.43 0.03 0.00 −0.09 0.07 IVD 3 0.31 0.50 0.060.14 0.05 0.29 −0.11 −0.22 *VG 5 0.22 0.48 0.05 0.25 −0.04 0.27 −0.12−0.11 *VT 4 0.25 0.07 0.06 0.62 −0.01 −0.14 −0.11 0.26 Backgroundfrequency 0.26 0.21 0.17 0.36

In Table 1, the combinations of the amino acids that showed significantbase selectivity were mentioned. That is, these results mean that thePPR motifs having the amino acid species of the No. 1 amino acid, No. 4amino acid, and No. “ii” (-2) amino acid (“NSRs (1, 4, and ii)” in thetable) that provided a significant P value are PPR motifs that impartbase-selective binding ability, and a larger “positive” value obtainedafter the subtraction of the background means higher base selectivityfor the base. Among the No. 1 amino acid, No. 4 amino acid, and No. “ii”(-2) amino acid, the No. 4 amino acid most strongly affects the baseselectivity, the No. “ii” (-2) amino acid affects the base selectivitynext strongly, and the No. 1 amino acid most weakly affects the baseselectivity among the three amino acids.

Example 2: Evaluation of Sequence-Specific DNA-Binding Ability PPRMolecules

In this example, artificial transcription factors were prepared byfusing VP64, which is a transcription activation domain, to the threekinds of DNA-binding type (expectedly) PPR molecules, p63, pTac2, andGUN1, and by examining whether they could activate luciferase reporterseach having a corresponding target sequence in a human cultured cell,whether the PPR molecules had a sequence-specific DNA-binding ability ornot was determined (FIG. 5).

Experimental Method

1. Preparation of PPR-VP64 expression vector

Only the parts corresponding to the PPR motifs in the coding sequencesof p63, pTac2, and GUN1 were prepared by artificial synthesis. For theDNA synthesis, the artificial gene synthesis service of Biomatik wasused. The pCS2P vector having the CMV promoter was used as a backbonevector, and each synthesized PPR sequence was inserted into it. Further,the Flag tag and nuclear transfer signal were inserted at the N-terminusof the PPR sequence, and the VP64 sequence was inserted at theC-terminus of the same. The produced sequences of p63-VP64, pTac2-VP64,and GUN1-VP64 are shown in Sequence Listing as SEQ ID NOS: 7 to 9.

2. Preparation of Reporter Vector Having PPR Target Sequence

A reporter vector (pminCMV-luc2, SEQ ID NO: 10) was prepared, in whichthe firefly luciferase gene was ligated downstream from the Minimal CMVpromoter, and a multi-cloning site was placed upstream of the promoter.The predicted target sequence of each PPR was inserted into the vectorat the multi-cloning site. The target sequence of each PPR (TCTATCACTfor p63, AACTTTCGTCACTCA for pTac2, and AATTTGTCGAT for GUN1, SEQ IDNOS: 11 to 13 in Sequence Listing) was determined by predicting themotif-DNA recognition codes of DNA-binding type PPR from the motif-RNArecognition codes observed in the RNA-binding type PPR. For each PPR,sequences containing 4 or 8 of target sequences were prepared, and usedin the following assay. The nucleotide sequences of the vectors areshown as SEQ ID NOS: 14 to 19 in Sequence Listing.

3. Transfection into HEK293 T Cell

The PPR-VP64 expression vector prepared in the section 1, the fireflyluciferase expression vector prepared in the section 2, and the pRL-CMVvector (expression vector for Renilla luciferase, Promega) as areference were introduced by using Lipofectamine LTX (LifeTechnologies). The DMEM medium (25 μl) was added to each well of a96-well plate, and a mixture containing the PPR-VP64 expression vector(400 ng), firefly luciferase expression vector (100 ng), and pRL-CMVvector (20 ng) was further added. Then, a mixture of the DMEM medium (25μl) and Lipofectamine LTX (0.7 μl) was added to each well, the plate wasleft standing at room temperature for 30 minutes, then 6×10⁴ of theHEK293 T cells suspended in the DMEM medium containing 15% fetal bovineserum (100 μl) were added, and the cells were cultured at 37° C. in aCO2 incubator for 24 hours.

4. Luciferase Assay

Luciferase assay was performed by using Dual-Glo Luciferase Assay System(Promega) in accordance with the instructions attached to the kit. Forthe measurement of the luciferase activity, Tri Star LB 941 Plate Reader(Berthold) was used.

(Results and Discussion)

The luciferase activity was compared for the cases of introducingpTac2-VP64 or GUN1-VP64 together with pminCMV-luc2 for a negativecontrol, or the reporter vector having 4 or 8 target sequences (tablementioned below, FIG. 6). The comparison of the activity was performedon the basis of standardized scores obtained by dividing the measuredvalues obtained with Fluc (firefly luciferase) with the measured valueobtained with Rluc (Renilla luciferase) as the reference (Fluc/Rluc). Asa result, there was observed a tendency that the activity increased withincrease of the number of the target sequence for the both cases, andthus it was verified that each of the PPR-VP64 molecules specificallybound to each target sequence, and functioned as a site-specifictranscription activator.

Fluc reporter PPR-VP64 Reference Fluc Rluc Fluc/Rluc Fold activationpTac2-VP64 (negative control) pminCMV-luc2 pTac2-VP64 pRL-CMV  477444948  9.649151172 1 pTac2-VP64 (4x target) pTac2-4x target pTac2-VP64pRL-CMV 133465 4757 28.05654824 2.907670089 pTac2-VP64 (8x target)pTac2-8x target pTac2-VP64 pRL-CMV 189146 4011 47.15681875 4.887146849GUN1-VP64 (negative control) pminCMV-luc2 GUN1-VP64 pRL-CMV  29590 3799 7.788891814 1 GUN1-VP64 (4x target) GUN1-4x target GUN1-VP64 pRL-CMV 61070 2727 22.39457279 2.875193715 GUN1-VP64 (8x target) GUN1-8x targetGUN1-VP64 pRL-CMV  66982 2731 24.52654705 3.14891356

What is claimed is:
 1. A method for modifying a genetic substance of acell, the method comprising: designing a DNA binding protein;determining a DNA base sequence coding for an amino acid sequence of afused protein comprising a functional region and a DNA binding regionconsisting of a DNA-binding protein; cloning said DNA base sequence;preparing a vector carrying said DNA base sequence and a cell containinga DNA having at least 9 target DNA bases as a target DNA base sequence;and introducing the vector into the cell so that the DNA binding regionof the fused protein binds to the DNA having the at least 9 target DNAbases, and therefore the functional region modifies the DNA having theat least 9 target DNA bases, wherein the DNA-binding protein contains atleast 9 PPR motifs for binding to the at least 9 target DNA bases,respectively, and having a structure of the following formula 1:(Helix A)-X-(Helix B)-L  (Formula 1) (wherein, in the formula 1: Helix Ais a part that can form an α-helix structure; X does not exist, or is apart consisting of 1 to 9 amino acids; Helix B is a part that can forman α-helix structure; and L is a part consisting of 2 to 7 amino acids),wherein, position numbers of amino acids in the PPR motifs are the sameas PF01535 in Pfam under the following definitions: the first amino acidof Helix A is referred to as Number 1 amino acid (Number 1 AA), thefourth amino acid as Number 4 amino acid (Number 4 AA), and when a nextPPR motif (M_(n+1)) contiguously exists on the C-terminus side of thePPR motif (M_(n)) (when there is no amino acid insertion between the PPRmotifs), the -2nd amino acid counted from the end (C-terminus side) ofthe amino acids constituting the PPR motif (M_(n)); when a non-PPR motifconsisting of 1 to 20 amino acids exists between the PPR motif (M_(n))and the next PPR motif (M_(n+1)) on the C-terminus side, the amino acidlocating upstream of the first amino acid of the next PPR motif(M_(n+1)) by 2 positions, i.e., the -2nd amino acid; or when any nextPPR motif (M_(n+1)) does not exist on the C-terminus side of the PPRmotif (M_(n)), or 21 or more amino acids constituting a non-PPR motifexist between the PPR motif (M_(n)) and the next PPR motif (M_(n+1)) onthe C-terminus side, the 2nd amino acid counted from the end (C-terminusside) of the amino acids constituting the PPR motif (M_(n)) is referredto as Number “ii” (-2) amino acid (Number “ii” (-2) AA), each PPR motif(M_(n)) contained in the DNA-binding protein is a PPR motif having aspecific combination of amino acids as the three amino acids of Number 1AA, Number 4 AA, and Number “ii” (-2) AA, such that the DNA-bindingprotein has at least 9 combinations of the three amino acidscorresponding to the respective target DNA bases of the target DNA basesequence, and each combination of the three amino acids is determinedaccording to any one of the following definitions: (2-1) when the targetDNA base to which the PPR motif binds is the three amino acids, Number 1AA, Number 4 AA, and Number “ii” (-2) AA are an arbitrary amino acid,glycine, and aspartic acid, respectively; (2-2) when the target DNA baseto which the PPR motif binds is the three amino acids, Number 1 AA,Number 4 AA, and Number “ii” (-2) AA, are glutamic acid, glycine, andaspartic acid, respectively; (2-3) when the target DNA base to which thePPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA,and Number “ii” (-2) AA, are an arbitrary amino acid, glycine, andasparagine, respectively; (2-4) when the target DNA base to which thePPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA,and Number “ii” (-2) AA, are glutamic acid, glycine, and asparagine,respectively; (2-5) when the target DNA base to which the PPR motifbinds is A, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, glycine, and serine,respectively; (2-7) when the target DNA base to which the PPR motifbinds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, isoleucine, and asparagine,respectively; (2-12) when the target DNA base to which the PPR motifbinds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, methionine, and asparticacid, respectively; (2-13) when the target DNA base to which the PPRmotif binds is C, the three amino acids, Number 1 AA, Number 4 AA, andNumber “ii” (-2) AA, are isoleucine, methionine, and aspartic acid,respectively; (2-15) when the target DNA base to which the PPR motifbinds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, asparagine, and asparticacid, respectively; (2-16) when the target DNA base to which the PPRmotif binds is T, the three amino acids, Number 1 AA, Number 4 AA, andNumber “ii” (-2) AA, are phenylalanine, asparagine, and aspartic acid,respectively; (2-17) when the target DNA base to which the PPR motifbinds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are glycine, asparagine, and aspartic acid, respectively;(2-18) when the target DNA base to which the PPR motif binds is T, thethree amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA,are isoleucine, asparagine, and aspartic acid, respectively; (2-19) whenthe target DNA base to which the PPR motif binds is T, the three aminoacids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are threonine,asparagine, and aspartic acid, respectively; (2-20) when the target DNAbase to which the PPR motif binds is T, the three amino acids, Number 1AA, Number 4 AA, and Number “ii” (-2) AA are valine, asparagine, andaspartic acid, respectively; (2-21) when the target DNA base to whichthe PPR motif binds is T, the three amino acids, Number 1 AA, Number 4AA, and Number “ii” (-2) AA are tyrosine, asparagine, and aspartic acid,respectively; (2-22) when the target DNA base to which the PPR motifbinds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, asparagine, and asparagine,respectively; (2-23) when the target DNA base to which the PPR motifbinds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are isoleucine, asparagine, and asparagine, respectively;(2-24) when the target DNA base to which the PPR motif binds is C, thethree amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA,are serine, asparagine, and asparagine, respectively; (2-25) when thetarget DNA base to which the PPR motif binds is C, the three aminoacids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine,asparagine, and asparagine, respectively; (2-26) when the target DNAbase to which the PPR motif binds is C, the three amino acids, Number 1AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid,asparagine, and serine, respectively; (2-27) when the target DNA base towhich the PPR motif binds is C, the three amino acids, Number 1 AA,Number 4 AA, and Number “ii” (-2) AA, are valine, asparagine, andserine, respectively; (2-28) when the target DNA base to which the PPRmotif binds is C, the three amino acids, Number 1 AA, Number 4 AA, andNumber “ii” (-2) AA, are an arbitrary amino acid, asparagine, andthreonine, respectively; (2-29) when the target DNA base to which thePPR motif binds is C, the three amino acids, Number 1 AA, Number 4 AA,and Number “ii” (-2) AA, are valine, asparagine, and threonine,respectively; (2-30) when the target DNA base to which the PPR motifbinds is C, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, asparagine, and tryptophan,respectively; (2-31) when the target DNA base to which the PPR motifbinds is T, the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are isoleucine, asparagine, and tryptophan, respectively;(2-33) when the target DNA base to which the PPR motif binds is T, thethree amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA,are an arbitrary amino acid, proline, and aspartic acid, respectively;(2-34) when the target DNA base to which the PPR motif binds is T, thethree amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA,are phenylalanine, proline, and aspartic acid, respectively; (2-35) whenthe target DNA base to which the PPR motif binds is T, the three aminoacids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are tyrosine,proline, and aspartic acid, respectively; (2-37) when the target DNAbase to which the PPR motif binds is A, the three amino acids, Number 1AA, Number 4 AA, and Number “ii” (-2) AA, are an arbitrary amino acid,serine, and asparagine, respectively; (2-38) when the target DNA base towhich the PPR motif binds is A, the three amino acids, Number 1 AA,Number 4 AA, and Number “ii” (-2) AA, are phenylalanine, serine, andasparagine, respectively; (2-39) when the target DNA base to which thePPR motif binds is A, the three amino acids, Number 1 AA, Number 4 AA,and Number “ii” (-2) AA, are valine, serine, and asparagine,respectively; (2-41) when the target DNA base to which the PPR motifbinds is the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are an arbitrary amino acid, threonine, and aspartic acid,respectively; (2-42) when the target DNA base to which the PPR motifbinds is the three amino acids, Number 1 AA, Number 4 AA, and Number“ii” (-2) AA, are valine, threonine, and aspartic acid, respectively;(2-43) when the target DNA base to which the PPR motif binds is A, thethree amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA,are an arbitrary amino acid, threonine, and asparagine, respectively;(2-44) when the target DNA base to which the PPR motif binds is A, thethree amino acids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA,are phenylalanine, threonine, and asparagine, respectively; (2-45) whenthe target DNA base to which the PPR motif binds is A, the three aminoacids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, areisoleucine, threonine, and asparagine, respectively; (2-46) when thetarget DNA base to which the PPR motif binds is A, the three aminoacids, Number 1 AA, Number 4 AA, and Number “ii” (-2) AA, are valine,threonine, and asparagine, respectively; (2-48) when the target DNA baseto which the PPR motif binds is C, the three amino acids, Number 1 AA,Number 4 AA, and Number “ii” (-2) AA, are isoleucine, valine, andaspartic acid, respectively; (2-49) when the target DNA base to whichthe PPR motif binds is C, the three amino acids, Number 1 AA, Number 4AA, and Number “ii” (-2) AA, are an arbitrary amino acid, valine, andglycine, respectively; and (2-50) when the target DNA base to which thePPR motif binds is T, the three amino acids, Number 1 AA, Number 4 AA,and Number “ii” (-2) AA, are an arbitrary amino acid, valine, andthreonine, respectively.
 2. The method according to claim 1, wherein thefunctional region is fused to the DNA-binding protein on the C-terminusside of the protein.
 3. The method according to claim 1, wherein thefunctional region is a DNA-cleaving enzyme, or a nuclease domainthereof, or a transcription control domain, and the complex functions asa target sequence-specific DNA-cleaving enzyme or transcription controlfactor.
 4. The method according to claim 3, wherein the DNA-cleavingenzyme is the nuclease domain of FokI (SEQ ID NO: 6).
 5. The methodaccording to claim 1, wherein the one or more PPR motifs are any groupof motifs selected from 9 PPR motifs belonging to the p63 proteinconsisting of the amino acid sequence of SEQ ID NO: 1, 11 PPR motifsbelonging to the GUN1 protein consisting of the amino acid sequence ofSEQ ID NO: 2, 15 PPR motifs belonging to the pTac2 protein consisting ofthe amino acid sequence of SEQ ID NO: 3, 10 PPR motifs belonging to theDG1 protein consisting of the amino acid sequence of SEQ ID NO: 4, and11 PPR motifs belonging to the GRP23 protein consisting of the aminoacid sequence of SEQ ID NO: 5.